Use of additives for the reduction of non-specific binding in assays

ABSTRACT

A method for reducing non-specific binding in an assay is provided herein. The method includes (a) providing a reaction mixture, which includes or is suspected to include a first component and a second component capable of binding to each other in a specific binding reaction, and (b) adding non-physiological amounts of at least one additive to the reaction mixture before, during or after binding in a sufficient amount to reduce non-specific binding in the reaction mixture. The method further includes (c) monitoring or measuring the presence and/or concentration of at least one of the first and second components after step (b).

FIELD OF THE INVENTION

The present invention relates to methods for reducing non-specificbinding in assays for the detection and/or measurement of an analyte ina sample. In particular, the invention relates to the use of additivesto reduce non-specific binding in assays.

BACKGROUND OF THE INVENTION

Fluorescence polarization and fluorescence intensity measurementsprovide a powerful means by which macromolecular association reactionscan be studied. These fluorescent techniques have been applied to studyantigen-antibody, hapten-antihapten, protein-ligand, and protein-DNAinteractions.

The inherent sensitivity of fluorescence measurements can be used inmonitoring the extent of reaction as a fluorescent reactant, F, combineswith its macromolecular partner, R:

where k₁ is the forward reaction and k⁻¹ is the back reaction such that(k₁)/(k⁻¹)=K_((eq)).

The investigator can choose to follow changes in the fluorescencepolarization (FP) and/or the fluorescence intensity (FI). If thereactants do not have natural fluorescence, as in the case of manyantigen-antibody systems, one of the reactants can be covalently labeledwith a fluorescent tag. An increase in the fluorescence polarization ofF usually occurs during combination with R, even if there are noconcomitant changes in the fluorescence intensity. This is because thepolarization increase reflects a slowing down of the rotary motion ofthe smaller ligand, F, when it becomes attached to the larger species,R. R is in many instances an antibody or a fragment of an antibody, suchas an F_(ab) or F_(ab2) (dimer). Equilibrium fluorescence polarizationand intensity measurements can be determined in a direct readoutpolarometer capable of measuring both the degree of fluorescencepolarization and the fluorescence intensity of a solution.

Immunoassays have been used in an effort to improve upon the success indetecting substances at very low levels. For example, the use of suchtechniques has been prompted by the extraordinary successes that havebeen achieved in the measurement of biological substances by specificimmunological reagents and techniques. Available evidence indicates thatspecific antibodies can be obtained against even low molecular weightorganic compounds, such as pesticides or other haptens.

Any means of applying an immunochemical reaction to a detection problemultimately relies upon a reaction occurring between a substance (antigenor hapten) and its specific antibody. One means by which thisinteraction can be employed in measurement and detection has come to beknown as “competitive binding assay”. In principle, this method requirestwo reagents. These are a labeled form of the substance to be detectedor measured, and an antibody or receptor specifically directed againstthe substance. The principle of the assay involves a preliminarymeasurement of the binding of the labeled hapten or antigen (substancebeing detected) with its antibody and then, a determination of theextent of the inhibition of this binding by known quantities of theunlabeled hapten or antigen, which corresponds to the unknown. Fromthese data, a standard curve can be constructed which shows the degreeof binding by the labeled hapten or antigen under certain specifiedconditions as a function of concentration of the unlabeled hapten orantigen or unknown added.

One way of implementing an immunoassay is to employ a fluorescent label.Usually, fluorescent labeling of one of the reagents e.g. the hapten isimportant in carrying out of the immunoassay by means of fluorescencepolarization and/or fluorescence intensity measurements. Unlike otherimmunoassays, such as ELISA, no physical separation of bound from freeforms of the labeled hapten is necessary. Therefore a simple rapidoptical measurement yields the essential information without physicalseparation of bound and free labeled materials.

One problem associated with immunoassays, as well as other assays, hasbeen non-specific binding. Ideally, in an immunoassay, the investigatorwants to follow a simple biomolecular reaction occurring between alabeled substance (antigen or hapten) and its specific antibody.However, the investigator must often contend with non-specific binding,such as antibody-antibody interactions, antigen-antigen interaction,hapten-hapten interaction, or interactions between the antibody orantigen (or hapten) and interfering substances in the assay. Suchnon-specific binding can make it extremely difficult, if not impossibleto measure specific binding reactions, especially when the equilibriumand rate constants for non-specific binding reactions are a significantfraction of those of the specific binding reactions.

Therefore, there is a need to provide improved and more sensitive assaysfor detecting the presence and/or amount of an analyte in a sample. Inparticular, it would be advantageous to provide competitive andnon-competitive assays in which non-specific binding in the assays issubstantially reduced. This would allow, for example, the investigatorto successfully follow the specific binding of a labeled antigen orhapten (substance being detected) with its antibody, an unlabeledantibody with a tracer, an unlabeled analyte with a labeled antibody,and many other analytes or labeled tracers with an appropriate “bindingpartner”, such as a fragment of an antibody, a receptor, or otherproteins.

SUMMARY OF THE INVENTION

The present invention is directed to methods for reducing non-specificbinding in assays which rely upon a reaction occurring between asubstance and its specific binding partner. In some embodiments, themethod involves providing a reaction mixture, which includes or issuspected to include a first component and a second component capable ofbinding to each other in a specific binding reaction. The method furtherincludes adding non-physiological amounts of at least one additive tothe reaction mixture before, during or after binding in a sufficientamount to reduce non-specific binding in the reaction mixture. Moreover,the method includes monitoring or measuring the presence and/orconcentration of at least one of the first and second components in thepresence of the at least one additive. If the first and secondcomponents do not have natural fluorescence, one of the first and secondcomponents can be covalently labeled with a fluorescent tag.

In some embodiments, the present invention provides competitive bindingfluorescence assays. In particular, the invention provides a method forreducing non-specific binding in a competitive-type fluorescence assayfor the detection and/or measurement of an analyte in a sample. Thismethod employs various reagents. For example, the method includesproviding a fluorescent conjugate of an analyte of interest; andproviding a component that specifically binds to the analyte and itsfluorescent conjugate. The method further includes combining thefluorescent conjugate and the specific binding component with a samplewhich includes or is suspected to include the analyte under conditionssuitable for the specific binding component to specifically bind to theanalyte and its fluorescent conjugate. Importantly, the method alsoincludes adding non-physiological amounts of at least one additive tothe sample before, during or after the combining step to minimizenon-specific binding in the assay. Significantly, the method includesmonitoring for the inhibition of the binding of the fluorescentconjugate to the specific binding component by the sample.

In the presence of the unlabeled analyte, a smaller percentage of thelabeled analyte is bound to the specific binding component. A standardcurve can be constructed from this type of data, which would showfluorescence measurements for certain standard chosen experimentalconditions plotted as a function of the amount of unlabeled analyte. Anunknown amount of analyte in the sample can then be determined from thisstandard curve.

The present invention further provides a kit for detecting and/ormeasuring an analyte of interest in a sample. The kit includes a labeledconjugate of the analyte; and an antibody or receptor that specificallybinds to the analyte. The kit also includes at least one additive. Thekit may optionally also include an unlabeled form of the analyte, whichis useful for constructing a standard curve.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph of the results of competitive inhibition of thebinding of F-thyroxine to its specific antibody by unlabeled thyroxine(T4).

FIG. 2 is a graph of the effects of an additive on specific andnon-specific binding.

FIG. 3 is a graph of a kinetic curve measurable in the presence of anadditive using a rhodamine-labeled thyroxine/anti-thyroxine system.

FIG. 4 is a graph and table of the influence of sodium benzoate combinedwith a solvent on the reaction rate of a labeled thyroxine withanti-thyroxine and on the non-specific binding.

FIG. 5 is a graph of the effects on a fluoresecein-T4/anti-T4 reactionperformed in a PBS buffer containing an 5% 2-propanol and differingamounts of sodium benzoate in the absence of non-specific bindingsubstances.

FIG. 6 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of sodium benzoate combined with 5%2-propanol in the presence of the non-specific binding substance BSA.

FIG. 7 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of guanidine hydrochloride combined with5% 2-propanol.

FIG. 8 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of sodium dichloroacetate combined with5% 2-propanol.

FIG. 9 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of potassium benzoate combined with 4.75%2-propanol.

FIG. 10 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of sodium chloroacetate combined with4.75% 2-propanol.

FIG. 11 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of sodium dichloroacetate combined with4.75% 2-propanol.

FIG. 12 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of sodium salicylate combined with 4.75%2-propanol.

FIG. 13 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of N-hydroxsuccinimide combined with4.75% 2-propanol.

FIG. 14 is a graph of the effects on a fluorescein-T4/anti-T4 systemusing differing concentrations of sodium chloride combined with 4.75%2-propanol.

FIG. 15 is a graph of the effects on a rhodamine-T4/anti-T4 system usingdiffering concentrations of sodium benzoate combined with 10%2-propanol.

FIG. 16 is a graph of a Standard Calibration Curve of arhodamine-T4/anti-T4 in the presence of sodium benzoate combined with10% 2-propanol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved assay by which macromolecularassociation reactions are studied. The improvement to previoustechnology is that non-physiological amounts of a one or more additivesselected from salts, alcohols, solvents or combinations thereof areadded to a reaction mixture in a sufficient amount to minimize or reducethe non-specific binding, as defined herein, in the reaction mixturesuch that the assay is easier to perform, easier to interpret, easier tocarry out successfully and improves results giving increased accuracy.The term “additive” as used herein is intended to include, but is notlimited to, chaotropes, cosmotropes, salting-in and salting-out agentsas outlined by the Hofmeister series¹, organic salts, inorganic salts,non-ionic organic compounds, organic solvents and/or combinationsthereof. ¹Hofmeister F: Zur Lehre von der Wirkung der Salze. Arch ExpPathol Pharmakol 1888, 24:247-260.

The reaction mixture includes a first component and a second componentcapable of binding to each other in a specific binding reaction. The useof at least one additive in non-physiological amounts reducesnon-specific binding, i.e. undesired binding or cross-reactivity,thereby allowing the investigator to follow a simple biomolecularreaction occurring between the first and second components. If thereactants (i.e., first and second components) do not have naturalfluorescence, as in the case of many antigen-antibody or hapten-antibodysystems, one of the reactants can be labeled with a fluorescent-tag.

The term “non-specific binding” as defined herein is the binding and orcross-reactivity of the first and/or second components in the reactionmixture to anything other than each other. In some embodiments, the atleast one additive reduces non-specific binding between molecules of thefirst component. In other embodiments, the at least one additive reducesnon-specific binding between molecules of the second component. In stillother embodiments, the at least one additive reduces or minimizesnon-specific binding and or cross reactivity occurring between the firstor second component and an interfering substance in the reactionmixture. Examples of interfering or cross-reacting substances include avariety of binders, such as bovine serum albumin (BSA) andimmunoglobulins, which may or may not have a definable relationship withthe analyte; or the walls of a container in which the analyte ismeasured; or other random biomolecules that have no definablerelationship with the analyte of interest.

It is understood that the additive or additives added to the reactionmay reduce all binding to a certain extent. However, it has beenobserved that the additive or combinations of additives are capable ofminimizing non-specific binding relative to specific binding. Seeingthat the intended specific binding may be slightly affected by theadditive, this side effect is minimal in comparison to the reduction ofthe non-specific binding caused by the additive, which permits moreaccurate and more precise measurements of the intended specific binding.In other words, this minimization of non-specific reactions or bindingcan occur at an extent equal to or much greater than the minimization ofthe specific binding. This leads to the ability to measure specificbinding free of interfering non-specific interactions.

In one embodiment, the assay of the present invention takes advantage ofthe well-known highly specific binding of antibodies to theircorresponding haptens or antigens. As mentioned above, non-specificbinding is a problem often associated with immunoassays. For example,antigen-antigen (hapten-hapten) and antibody-antibody interactions canoccur. Moreover, the antibody can form immune complexes with substancesother than its specific antigen or hapten, which may be present in theassay. In particular, an antibody only recognizes a small part of theantigen molecule, the so-called epitope. Any molecule containing such anepitope accessible for the antibody, will bind as if it were the analyteof interest. The impact is largely dependent on the relative affinity tothe antibody and the relative concentration of the antibody molecule incomparison with the affinity and concentration of the analyte (i.e., theantigen or hapten).

Before the present invention, the cross-reactivity in antigen-antibodyor hapten-antibody binding of structural analogues could not always becontrolled in a manner that only a single analyte would react with theantibody. The improvement in the assay of the present invention includesconducting an assay, such as an immunoassay, in the presence ofnon-physiological amounts of at least one additive. This reducesnon-specific binding without disturbing the specific immunochemicalreaction, relative to specific binding.

In one embodiment, the at least one additive is present in the assay inan amount of about 0.2 M to about 2.5 M or higher. In anotherembodiment, the at least one additive is present in an amount of about5% to about 20% (weight/volume) of the reaction mixture.

Suitable additives include salts of an anion selected from thefollowing: chloride, bromide, iodide, salicylate, trichloroacetate,thiocyanate, perchlorate and benzoate. In some embodiments, the additiveis selected from the following agents: 8-anilino-1-napthalene-sulfonicacid, 2-Guanidinobenzimidazole, 2,3,5-triacetylguanosine,Benzimidazolylurea, acetamide,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), sodiumtrichloroacetate, sodium deoxycholate, creatine benzimidazole, sodiump-toluene-sulfonate, sodium dichloro acetate, sodium iodide, sodiumfluoride, sodium chloroacetate, 5-benzimidazolecarboxylic acid,Salicylamide, guanidine hydrochloride, sodium chloride,2-benzimidazole-proprionic acid, 2-benzimidazolemethanol, Sodiumchlorodifluoroacetate, 4-guanidinobenzoic acid, 3-chlorobenzoic acid,N-hydroxy succinimide, guanidine and Potassium benzoate, organicsolvents and combinations thereof. The present inventors have foundthat, when present in non-physiological amounts, these additives disruptnon-specific binding, but essentially do not disturb the specificbinding reaction. Non-physiological amounts of the salt agents includeranges of 0.2 M to 2.5 M, desirably 0.3 to 2.0 M, more desirably 0.4 to2.5 M and even more desirably 0.5 to 1.5 M. However, the upper limitsalt concentration may go beyond these ranges, provided that it does nothave deleterious effects on the assay and is largely dictated bypractical considerations, such as unwanted precipitation and/ordenaturation of reaction components, excessive precipitation of salts,general interference with the results, among other practicalconsiderations.

In one embodiment, the additive affects the order of reaction withrespect to the first component and the second component in the reactionmixture. In one preferred embodiment, the additive makes the bindingreaction first order with respect to the concentration of each of thefirst and second components. This allows the investigator to follow asimple second order reaction between the first and second components.This is described in further detail below.

In some embodiments, at least one of the first or second specificbinding components is fluorescently-labeled. Use of a fluorescent labelallows the methods of the present invention to be carried out either byfluorescence polarization measurements or, in some cases, byfluorescence intensity measurements. In some embodiments, a method ofthe present invention is selected from the following: a fluorescencepolarization assay, a fluorescence-based enzyme-linked immunosorbentassay (ELISA) and a Polarized Fluorescence Intensity Difference (PFID)assay. In one preferred embodiment, a method of the present invention isa fluorescence polarization assay.

As described above, an increase in fluorescence polarization or a changein fluorescence intensity via enhancement or quenching of a fluorescentreactant usually occurs during the combination with its macromolecularpartner. An increase in polarization reflects a slowing down of therotary motion of the fluorescent reactant when it becomes attached toits macromolecular partner. This increase in fluorescence polarizationoccurs even if there are no concomitant changes in the fluorescentintensity.

In the methods of the present invention, equilibrium fluorescencepolarization and intensity measurements can be made in a direct readout“polarometer”. Moreover, kinetic measurements of slow kinetic processes(10 seconds or greater) can also be made in a direct readoutpolarometer.

Polarometer denotes an instrument for measuring the degree ofpolarization as contrasted to optical rotation. The solution to bemeasured is first excited in a standard cell by linearly polarized lightof appropriate wavelength. The emission fluorescent beam (withappropriate filters) then passes through a rapidly rotating polarizerand onto a photomultiplier tube whose output is fed into a computerwhich calculates the fluorescence polarization, p=(V−H)/(V+H), PolarizedFluorescence Intensity Difference or PFID (V−H), and the totalfluorescence intensity, V+H. V and H denote intensities of verticallypolarized and horizontally polarized components in fluorescent light.Alternatively, a “T-format” polarometer using two photomultiplier tubesset at right angles to the excitation source and each having polarizingfilters place in a mutually orthogonal position. Provision is made forautomatic deduction of the blank. Temperature control of the cellcompartment is maintained with an appropriate thermostat.

Direct readout polarometers are available commercially. For example,such instruments are available from the following vendors: BMG LabtechGmbH, Offenburg, Germany; JASCO Corporation, Tokyo, Japan; Tecan SchweizAG, Hännedorf, Switzerland; Bioscan, Inc, Washington, D.C.; MolecularDevices Corporation, Sunnyvale, Calif.; Perkin Elmer Life and AnalyticalSciences, Inc., Wellesley, Mass.; Photon Technology International, Inc.,Birmingham, N.J.; Abbott GmbH & Co. KG, Wiesbaden, Germany; DiachemixCorp. (USA), Whitefishbay, Wis.; and Invitrogen Corp., Carlsbad, Calif.

As described above, the inherent sensitivity of fluorescencemeasurements can be used in monitoring the extent of reaction as afluorescent reactant, F, combines with its macromolecular partner, R:

where k₁ is the forward reaction and k⁻¹ is the back reaction such that(k₁)(k⁻¹)=K_((eq)).

The ratio of bound to free fluorescent material in Eq. (1) above can bedirectly related to fluorescence polarization and intensity parameters,as shown in the equations below (Dandliker, et al, (1969)Immunochemistry, 6, 125):

$\begin{matrix}{\frac{F_{b}}{F_{f}} = {\frac{Q_{f}}{Q_{b}}\left( \frac{P - P_{f}}{P_{b} - P} \right)}} & {{Eq}.\mspace{14mu}(2)} \\{\frac{F_{b}}{F_{f}} = \frac{Q_{f} - Q}{Q - Q_{b}}} & {{Eq}.\mspace{14mu}(3)}\end{matrix}$

In equations (2) and (3) above, the symbols or subscripts are asfollows: f and b, denote free and bound forms, respectively; p denotesthe polarization of fluorescence; F denotes fluorescent-labeledmaterial; and Q denotes the ratio of fluorescence intensity to molarconcentration of fluorescent-labeled material.

If the binding sites on the fluorescent reactant's macromolecularpartner are uniform, the results can be treated by the Scatchard form ofthe mass law,

$\begin{matrix}{\frac{F_{b}}{F_{f}} = {K\left( {F_{b,\max} - F_{b}} \right)}} & {{Eq}.\mspace{14mu}(4)}\end{matrix}$where F_(b,max) is the maximum value of F_(b) and is taken to be equalto the initial binding site concentration of R. R in equation 4 is equalto (F_(b,max)−F_(b)). K is the association constant for the reaction andthe sum, F_(b)+F_(f)=M, is the known concentration of fluorescentreactant.

The interpretation of kinetics in terms of fluorescence polarization wascarried out by initial rate equations:

$\begin{matrix}{\left( \frac{\mathbb{d}F_{b}}{\mathbb{d}t} \right)_{0} = {{- \left( \frac{\mathbb{d}F_{f}}{\mathbb{d}t} \right)_{0}} = {\frac{Q_{f}}{Q_{b}}\left( \frac{F_{f,0}}{P_{b} - P_{f}} \right)\left( \frac{\mathbb{d}P}{\mathbb{d}t} \right)_{0}}}} & {{Eq}.\mspace{14mu}(5)} \\{\left( \frac{\mathbb{d}P}{\mathbb{d}t} \right)_{0} = {\frac{Q_{b}}{Q_{f}}\left( {P_{b} - P_{f}} \right){k\left( F_{b,\;\max} \right)}\left( N_{1} \right)\left( F_{f,0} \right)\left( {N_{2} - 1} \right)}} & {{Eq}.\mspace{14mu}(6)}\end{matrix}$where k is the usual second order rate constant. For constant F_(b,max)but varying (F_(f))_(o)

$\begin{matrix}{{\log\left( \frac{\mathbb{d}P}{\mathbb{d}t} \right)}_{0} = {{\left( {N_{2} - 1} \right){\log\left( F_{f,0} \right)}} + {constant}}} & {{Eq}.\mspace{14mu}(7)}\end{matrix}$Alternatively, for constant (F_(f,o)), but varying (F_(b,max)):

$\begin{matrix}{{\log\left( \frac{\mathbb{d}P}{\mathbb{d}t} \right)}_{0} = {{\left( N_{1} \right){\log\left( F_{b,\max} \right)}} + {constant}}} & {{Eq}.\mspace{14mu}(8)}\end{matrix}$

In the kinetic equations, P is the value of the polarization, N₂ is theorder of the reaction with respect to the fluorescent reactantconcentration, and N₁ that with respect to the concentration of bindingsites on its macromolecular partner. The subscript “0” refers to zerotime. Equations (7) and (8) are especially useful in determining theorder of a reaction, which is an important characteristic to establishwhen investigating kinetic relationships. If the order with respect tothe concentration of each reactant proves to be constant over a wideconcentration range, then it is likely that the path of the reaction isalso remaining the same which gives some assurance that derived kineticconstants have some simple physical meaning. Also, equations (7) and (8)are in a form easy to use, since it is not necessary to know theabsolute, but only the relative concentrations of each reactant. Dealingwith initial rates while focusing only upon the initial stages ofreaction accomplishes some simplification by avoiding the back reactionand the inhibition by product.

In some embodiments of the present invention, a non-physiological amountof a suitable additive affects the order of the reaction with respect toa fluorescently-labeled reactant (e.g., fluorescently-labeled analyte)and its macromolecular partner (e.g., antibody). In one example, theadditive makes the binding reaction first order with respect to theconcentration of each of the fluorescent reactant and its macromolecularpartner (i.e., N₁ and N₂ will each be 1).

As described above, there are situations where fluorescence polarizationand intensity measurements can not be easily made. For example, p, V−Hand V+H measurements are difficult if not impossible in situations wherethere is non-specific binding occurring in the reaction mixture. This isdescribed further in the examples below. The present invention isdirected to overcoming the problem of non-specific binding by employingnon-physiological amounts of at least one additive in the reactionmixture. Preferably, the additive is present in an amount of about 0.5 Mto about 1.5 M or higher.

In one embodiment, the method of the present invention is a competitiveinhibition-type assay. The method is useful for detecting and/ormeasuring unknowns. For example, the assay is useful for the detectionof a hapten or antigen in a sample. In one embodiment, the substance tobe detected is a low molecular weight organic contaminant of environmentconcern. Such contaminants may be in a food or soil sample, for example.In another embodiment, the substance to be detected is a biologicalsubstance in a patient sample, e.g., blood or serum.

In one embodiment, the method of the present invention is anon-competitive type assay where the presence or absence of a proteinmoiety (e.g., enzyme, antibody or receptor), an oligonucleotide, orother macromolecule is detected. The method is useful for detectingand/or measuring the concentration of the macromolecule. Furthermore,other parameters, such as the equilibrium and rate binding constants ofthe macromolecule with a partner of interest, can be determined.

In some other embodiments, a competitive inhibition type assay of thepresent invention employs a fluorescent conjugate of the substance(analyte) of interest, together with an antibody to the analyte. In someembodiments, the antibody is first exposed to the fluorescent-labeledform of the analyte. Then, a study of the inhibition of the binding ofthis fluorescent conjugate by a sample thought to contain the analyte ofinterest is performed. The assay is done in the presence ofnon-physiological amounts of at least one additive.

In particular, the invention provides a general method for reducingnon-specific binding in a fluorescence assay for the detection and/ormeasurement of an analyte in a sample. This method includes providing afluorescent conjugate of the analyte; and providing a component thatspecifically binds to the analyte and its fluorescent conjugate. Themethod further includes combining the fluorescent conjugate and thespecific binding component with a sample which includes or is suspectedto include the analyte under conditions suitable for the specificbinding component to specifically bind to the analyte and itsfluorescent conjugate. Moreover, the method includes addingnon-physiological amounts of at least one additive before, during orafter the combining step to reduce non-specific binding in the assay;and monitoring for the inhibition of the binding of the fluorescentconjugate to the specific binding component by the sample.

The monitoring step can include measuring the binding of the fluorescentconjugate to its specific binding component and determining the extentof inhibition of this binding by different known quantities of unlabeledanalyte. The monitoring step can also include constructing a standardcurve which shows the degree of binding by the fluorescent conjugate asa function of the quantity of the unlabeled analyte. The amount ofanalyte in the sample can then be determined by measuring the binding ofthe fluorescent conjugate to its specific binding component in thepresence of the sample and determining the amount of analyte in thesample from the standard curve.

One competitive-type assay of the present invention is preferably afluorescence polarization assay. Other assays include radioimmuneassays, fluorescence intensity assays, enzyme immunoassays (e.g.,ELISA), gold-colloidal assays, latex-bead assays, or any otherhomogeneous or heterogeneous type assays consisting of the bindingbetween a macromolecule and a binding partner. In one desiredembodiment, the monitoring step involves monitoring for a change in theinitial rate of polarized fluorescence intensity difference (PFID),defined as the absolute difference in the polarized fluorescence in thevertical and horizontal directions, i.e. (V−H), or d(V−H)/dT as afunction of an amount of analyte in the sample. For example, V−H (PFID)versus time is measured before and then after the sample is added, andthe total difference between these values over a set time period iscalculated. The total change in PFID is then compared to a standardcurve to determine the amount of analyte. Such a standard curve isconstructed by monitoring for a change in the initial rate ofpolarization as a function of different known quantities of unlabeledanalyte. The amount of analyte in the sample can be determined from thestandard curve.

Suitable additives to employ in a competitive-type assay of the presentinvention are the same as those described above. Preferably, theadditive is present in these assays in an amount of about 0.5 M to about1.5 M or higher, or from about 5% to about 20% w/v (of the assaymixture).

In one embodiment of the competitive assay, the additive affects theorder of reaction with respect to the analyte and the component thatspecifically binds to the analyte. For example, in one embodiment, theadditive makes N₁=1 in eq. 8 and N₂=1 in Eq. 7. That is, in oneembodiment, the additive is present in a sufficient amount to make thebinding reaction first order with respect to the concentration of eachof the analyte and its specific binding partner.

In one embodiment of the competitive assay, the additive reducesnon-specific binding between molecules of the analyte. In anotherembodiment, the additive reduces non-specific binding between moleculesof the analyte's specific binding partner (i.e., its specific bindingcomponent). In yet another embodiment, the additive reduces non-specificbinding between an interfering substance in the assay and at least oneof the analyte and its specific binding component.

The particular fluorescent moiety chosen to form the conjugate with theanalyte is selected from any number of fluorescent moieties. The choiceof fluorescent moiety is to a large extent a matter of convenience oncea coupling chemistry has been selected. Virtually any fluorophore havinga fluorescence lifetime of between 0.1 and 50 nanoseconds and having anexcitation wavelength of 350 to 800 nanometers is suitable for purposesof the present invention. For a detailed listing of fluorophores, whichare commercially available, see Handbook of Fluorescent Probes andResearch Chemicals, ed. Karen Larison, by Richard P. Haugland, Ph.D.,5^(th) ed., 1992, published by Molecular Probes, Inc. Some examples ofsuitable fluorescent moieties include the following: 7-AAD, AcridineOrange, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594,Aminonapthalene, Benzoxadiazole, BODIPY 493/504, BODIPY 505/515, BODIPY576/589, BODIPY FL, BODIPY TMR, BODIPY TR, Carboxytetramethylrhodamine,Cascade Blue, Coumarin, CY2, CY3, CY5, CY9, Dansyl Chloride, DAPI,Eosin, Erythrosin, Ethidium Homodimer II, Ethidium Bromide,Fluorescamine, Fluorescein, FTC, GFP (e.g. yellow shifted mutants T203Y,T203F, S65G/S72A), Hoechst 33242, Hoechst 33258, IAEDANS, Indopyras Dye,Lanthanide Chelate, Lanthanide Cryptate, Lissamine Rhodamie, LuciferYellow, MANT, MQAE, NBD, Oregon Green 488, Oregon Green 514, OregonGreen 500, Phycoerythrin, Porphyrin, Propidium Iodide, Pyrene, PyreneButyrate, Pyrene Maleimide, Pyridyloxazole, Rhodamine 123, Rhodamine 6G,Rhodamine Green, SPQ, Texas Red, TMRM, TOTO-1, TRITC, YOYO-1, VitaminB12, flavin-adenine dinucleotide, 6-carboxy-X-rhodamine,nicotinamide-adenine, and dinucleotide. Preferably, the fluorescentconjugate would have a fluorescent wavelength different from competingfluorescent substances which may occur in host samples of interest,e.g., blood serum, urine, tissue and extracts thereof.

In one embodiment, the analyte of interest is an organic contaminant.The organic contaminant can be one of environmental concern. Forexample, in one embodiment, the organic contaminant is a fungal ormicrobial toxin. Other analytes that can be detected with a method ofthe present invention include, but are not limited to, drugs, steroids,hormones, proteins, peptides, lipids, sugars, receptors, nuceic acids,vitamins, etc. For example, in one embodiment, the analyte of interestis thyroxine, which is a major hormone secreted by the follicular cellsof the thyroid gland.

In some embodiments, the component that specifically binds to theanalyte of interest is an antibody. It is well known that a substance,when injected into an animal, stimulates the animal to produce antibody.The antibody is capable of reacting with the injected substance in ahighly specific manner. These antibodies belong to a group of serumproteins known as immunoglobulins. The production of these antibodies asa result of the injection of the antigen takes place over a period ofmany weeks, and depends upon the immunization schedule. In general,“good” antigens are usually of large molecular size (greater than 20,000MW), partially digestible by enzymes and are recognized as being foreignby the antibody-producing animal.

Many compounds of environmental concern do not have a large molecularweight, and would, therefore, appear to be incapable of stimulatingantibody formation. However, this is not the case. So-called partialantigens or haptens can be produced and are capable of reacting withspecific antibody. Haptens or partial antigens are defined as antigenswhich alone cannot induce antibody formation, but in conjugation with asuitable carrier can produce antibody against themselves, as well asagainst the carrier-hapten complex. Examples of carriers includeovalbumin, bovine serum albumin, fibrinogen, and many others.Conjugation may be carried out by methods known in the art (Coligan, J.E. et al. (Eds.) Current Protocols in Immunology, Chapter 9, WileyIntersciences, New York, 1999).

The hapten, once conjugated with a suitable carrier, can stimulateantibody production. Some antibody will be produced which is highlyspecific in its reaction with the hapten alone. Therefore, by employinghapten-specific antibodies, the methods of the present invention can beused in the detection and quantitation of even low molecular weightorganic compounds, such as pesticides.

Antibodies suitable for use in the methods of the present inventioninclude polyclonal and monoclonal antibodies. Polyclonal antibodies canbe prepared in accordance with known methods (Coligan, J. E, et al.(Eds.), Current Protocols in Immunology, Wiley Intersciences, New York,1999).

Monoclonal antibodies may be produced by methods known in the art. Thesemethods include the immunological method described by Kohler andMilstein in Nature 256:495-497 (1975) and by Campbell in “MonoclonalAntibody Technology, The Production and Characterization of Rodent andHuman Hybridomas” in Burdon et al. (Eds.), Laboratory Techniques inBiochemistry and Molecular Biology, Volume 13, Elsevier SciencePublishers, Amsterdam (1985); and Coligan, J. E, et al. (Eds.), CurrentProtocols in Immunology, Wiley Intersciences, New York, (1999); as wellas the recombinant DNA method described by Huse et al., Science246:1275-1281 (1989).

Antibodies against markers in normal human tissue and neoplasms arecommercially available, for example, from Invitrogen Corporation(Carlsbad, Calif.), Advanced Immunochemical Inc. (Long Beach, Calif.)and RDI Division of Fitzgerald Industries, Intl. (formerly ResearchDiagnostics, Inc., Concord, Mass.). These include, but are not limitedto, monoclonal and polyclonal antibodies against the following classesof analytes: proteins (e.g., enzymes, growth factors, cytokines),peptides, receptors (e.g., CD markers), toxins, infectious agents (e.g.,viruses), steroids, hormones, lipids and lipoproteins). For example,antibodies against angiogenesis markers are commercially available, andsuch antibodies include those against the following receptors: CD31,CD34, Vascular Endothelial Growth Factor (VEGF), Vascular EndothelialGrowth Factor C (VEGF-C) and Vascular Endothelial Growth Factor Receptor3 (VEGFR-3). Also, antibodies against cancer markers are commerciallyavailable, such as those against the following: ALK, ALK 400, c-kit(CD117), COX-1, COX-2, EZH2, Ezrin, MAGE-A, Mesothelin, MTA1, NY-ESO-1,PDEF, PRAC, PSMA, RCAS1, thymidylate synthase and tyrosinase.Furthermore, antibodies against markers for specific types of cancer arecommercially available. For example, commercially available antibodiesagainst breast cancer markers include those against the following:BCA-225, Bcl-2, c-Met, Cathepsin D, CD63, cytokeratins, E-cadherin,EGFr, estrogen receptor, progesterone receptor, HER2, HER4, p53 andphospho-MAP kinase. Moreover, markers for colon cancer include, but arenot limited to, CA 19-9, CEA, COX-1, Ezrin, MLH1, MSH2, MSH6, PlateletDerived Endothelial Cell Growth Factor (PD-ECGF), PRLr and ThymidylateSynthase (TS). Antibodies against these colon cancer markers, as well asmarkers for other specific cancers, diseases or disorders are alsocommercially available.

Antibodies against apoptosis markers are also commercially available,and these markers include, for example, Bax, Bcl-2, Bcl-XL and PARP.Moreover, antibodies against cell cycle and cell proliferation markersare commercially available. Such cell cycle and cell proliferationmarkers include, but are not limited to, BrdU, Cyclin D1, Cyclin E, p21,Proliferating Cell Nuclear Antigen (PCNA) and S-Phase Kinase-AssociatedProtein 2 (SKP2). Antibodies against cellular proteins, such as, but notlimited to, calcitonin, HLA DR, MGMT, nitrotyrosine and nNOS are alsocommercially available. Moreover, antibodies against cytoskeletalproteins, such as α-Tubulin, β-Tubulin, Actin, Desmin, GFAP, Myosin,Ubiquitin and Vimentin, are commercially available. Furthermore,antibodies against markers for lipoprotein metabolism are commerciallyavailable; Such antibodies include, for example, those against HDL, LDL,APOE, ApoE2, LDL receptors, etc. The abbreviations used herein for themarkers are well known in the art.

Furthermore, antibodies against pesticides and herbicides arecommercially available from such companies as Guildhay, Ltd. (Guildford,Surrey, England). For example, antibodies against 2,4-D, aldrin,atrazine, chlortoluron, diuron, isoproturon, MCPA, mecoprop, paraquat,simazine, solanine, etc. are commercially available.

The present invention is not, in any way, limited to the specificexamples provided herein of analytes and antibodies that specificallybind to the analytes.

In other embodiments of the present invention, the component thatspecifically binds to the analyte is a receptor. For example, if ahormone carrying a fluorescent label attaches to its receptor, thehormone is thereby largely immobilized and this immobilization isregistered by an increase in the polarization of the emission from theattached fluorescent label.

In addition to antigen-antibody, hapten-antibody and hormone-receptorinteractions, the methods of the present invention can also be appliedto enzyme-substrate, protein-DNA, peptide-antibody and ligand-receptorinteractions.

Table A below provides some further examples of analytes, which may bedetected and/or measured using the methods of the present invention. Insome embodiments, a fluorescent conjugate of the analyte can be employedin methods of the present invention. The analysis may be performed, forexample, in water, serum, blood, urine or other bodily fluids. Moreover,the analysis may be performed in milk, wine, juices and food extracts.Table A is for illustrative purposes only and is not intended to limitthe scope of the present invention.

TABLE A CLASS EXAMPLES Pesticides Acetochlor and Other Acetanilides,atrazin, simazine, triazine, 2,4-D, 2,4,5-T, dichlorprop, MCPA, MCPB,Triclopyr, Pentachlorophenol, DDT, Isoproturon, Methabenzthiazurone,Metasulfuron-methyl, Chlorsulfuron, Acetochlor, Propanil, Paraquat,Parathion-methyl, BTEX, Nonylphenol, LAS Lipids DHET Sugars Bacterialsugars, polysaccharides Peptides, Proteins, Receptors IGG, Albumin,Receptors, KLH, LPH Myoglobin, Feto proteins, AT1 Oligonucleotides, DNA,Bacterial, animals, plants, specific sequences RNA Toxins Zearalenone,Deoxynivalenol, T-2, Aflatoxins, Ochratoxin, Fumonisins, Patulins,Trichothecene, citrinin, Cyclopiazonic acid, monoliformin,sterigmatocystin, alternaria-mycotoxins, ergot alkaloids Vitamins B 12,Vitamin C, Folic acid Small Molecules Chlorinated compounds, metalsTherapeutic drugs, e.g., Theophylline, doxorubicin, methotrexate,disopyramide, antiasmatics, antineoplastics, lidocaine, procainamide,propranolol, quinidine, N-acetyl- antiarythmics, procainamide,Phenobarbital, phenotoin, pridon, valproic anticonvulsants, antibiotics,acid carbamazepine, ethosuximide, cephalosporins, antiarthritics,antidepressants, erythromycin, tetracyclin, vancomycin, gentamicin, etc.amikacin, chloramphenicol, streptomycin, tobramycin, penicillin,salicylate, nortriptyline, amitriptoline, imipramine, desipramine,liodcaine, histamine, thyroxin, triiodothyronine, serotonin, etc. Drugsof abuse Morphine, heroin, hydromorphone, dihydrocodeine, pholcodine,Amphetamine, etc. Steroids and hormones Esterone, estradiiol, cortisol,testosterone, progesterone, chenodeoxycholic acid, digoxin, cholic acid,digitoxin, deoxycholic acid, lithocholic acid, etc. Prostaglandins PGE,PGF, PGA others Components of binding and Antibodies, receptors, enzymes(i.e., proteases, nucleases, reaction studies phosphatases)

In one embodiment of the present invention, the component thatspecifically binds to the analyte and its fluorescent reactant is on asolid substrate. For example, a specific binding component, such as anantibody, can be deposited on a glass, plastic or paper substrate.Substrates can include various microporous filters, such as PVDFfilters, nitrocellulose filters, cellulosic filters and the like. In oneexample, the antibody can first be bound to a substrate, such as PVDF.Second, the antibody on the substrate can be exposed to afluorescent-labeled form of an analyte of interest. Then, a study of theinhibition of the binding of this fluorescent conjugate by a samplethought to contain the analyte of interest is performed. In particular,one can observe a change in p, V−H and/or V+H which occurs in thepresence of the sample.

The competitive-inhibition assay of the present invention can be appliedto the simultaneous analysis of multiple analytes in a sample usinganalytes labeled with different fluorescent wavelength conjugates. Thiswould reduce the time and effort involved in multi-analyte, multi-sampleanalyses.

As described above, the present invention also provides a kit fordetecting and/or measuring an analyte of interest in a sample. The kitincludes the following components: a fluorescent conjugate of theanalyte; an antibody or receptor that specifically binds to the analyte;and at least one additive. The additive in the kit may be a salt of ananion selected from the following: chloride, salicylate,trichloroacetate, thiocyanate, perchlorate and benzoate. In oneembodiment, the kit further includes an unlabeled form of the analyte.

The following examples are for illustrative purposes only, and are notto be construed as limiting the present invention.

EXAMPLES Example 1 Materials

Preparation of Buffers

The influence of at least one additive on a reaction occurring betweenspecific binding molecules was investigated in pH 7.5 buffer solutions.These buffer solutions were denoted “Buffer 1” and “Buffer 2” and eachcontained a different amount of sodium benzoate.

Buffer 1:

0.5 M sodium benzoate in Phosphate Buffered Saline (PBS), adjusted to pH7.5. The final concentration of sodium benzoate in buffer 1 was about7.2% w/v.

Buffer 2:

1.0 M sodium benzoate in PBS, adjusted to pH 7.5. The finalconcentration of sodium benzoate in buffer 2 was about 14.4% w/v.

Preparation of Anti-Sera Stock Solution

An initial stock solution of anti-thyroxine sera was prepared. Inparticular, 1 ml of polyclonal anti-thyroxine sera containing 10 mg/mlIgG (Sigma) was diluted 1:5 in PBS buffer, pH 7.5. The finalconcentration of IgG in the stock solution was about 2 mg/ml.

Preparation of Thyroxine Stock Solution

An initial stock solution of an analyte (thyroxine) was prepared.Unlabeled thyroxine at 1 mg/ml in 0.5 M NaOH was diluted 100-fold inPBS. The final concentration of thyroxine in the stock solution wasabout 10 μg/ml.

Preparation of Fluorescein-Labeled Thyroxine Stock Solution

An initial stock solution of fluorescein-labeled analyte (F-thyroxine)was prepared. Fluorescein-labeled thyroxine at 1248 g/mol in alyophilized form was obtained from emp Biotech GmbH. First, 5 mg (4×10⁻⁶moles) of F-thyroxine was diluted to 10 ml in PBS to get a 4.1×10⁻⁴ Msolution of F-thyroxine. This solution was then further diluted 100-foldin PBS. The final concentration of F-thyroxine in the stock solution wasabout 4.1×10⁻⁶ M.

Example 2 Kinetic Curve Measurement Methodology

The following is a protocol useful for performing a fluoresceinpolarization assay of the present invention. All steps are performed atambient temperature. The total final reaction volume after all additionsis 3030 μl.

-   -   1. Add 20 μl or 0 μl (control) anti-sera stock solution to about        2980 μl of Buffer 1.    -   2. Pipette the diluted anti-sera solution prepared in step 1 up        and down from 1 to 10 seconds to thoroughly mix.    -   3. Add between 0 and 20 μl thyroxine stock solution (e.g., 0,        0.5, 2.0, 5.0 and 20 μl) to the mixed anti-sera from step 2.    -   4. Pipette the mixture formed in step 3 up and down for 1 to 10        seconds and then incubate for 30 to 300 seconds.    -   5. Add 10 μl of the Fluorescein-labeled thyroxine stock solution        to the incubated mixture from step 4.    -   6. Pipette the mixture formed in step 5 up and down for 1 to 10        seconds and begin measuring polarization (P), Polarized        Fluorescence Intensity Difference (V−H) or quenching (V+H total        intensity) versus time for between 10 and 7200 seconds after end        of mixing in a direct readout polarometer or between 0.1 and        7200 seconds after end of mixing with a stopped-flow        polarometer.    -   7. Determine the rate of reaction (initial rates) and plot        versus amount of thyroxine stock solution added in step 3 to        produce a standard curve.    -   8. Against a standard curve, an unknown amount of thyroxine can        be measured by comparison of initial rate measurement (speed of        reaction) of unknown with the standard curve.

Example 3 Kinetic Curve Measurable in the Presence of an Additive Usinga Fluorescein-Labeled Thyroxine/Anti-Thyroxine System

This example demonstrates that a reaction between F-thyroxine and itsspecific antibody was only capable of being followed when the reactionwas performed in the presence of an additive. The additive in thepresent example was sodium benzoate The thyroxine/anti-thyroxine systemis well known to be a very difficult system to measure due tothyroxine's capacity to bind to many things non-specifically. It was forthis reason that the thyroxine-anti-thyroxine system was chosen as amodel system to demonstrate the present invention.

The materials described in Example 1 and the protocol described inExample 2 were used for each of Experiments 1 to 5 shown in Table 1below, except that for Experiments 1 to 3, the reaction was performed inPBS instead of Buffer 1. Experiment No. 1 is a control to establishbaseline measurements for polarization (P), V−H, and V+H.

TABLE 1 Experiment Antiserum Thyroxine F-thyroxine No. (μl) (μl) (μl)Additive 1 0 0 10 − 2 20 0 10 − 3 20 20 10 − 4 20 0 10 + 5 20 20 10 +Results of Experiment 1

Polarization, V−H and V+H were measured versus time for between 10 and600 seconds. During this time period, fluorescence polarization baselinemeasurements were constant. In particular, V−H values were between 0.198to 0.193 V, V+H values were between 2.135 to 2.130, and V and P valueswere between 91 to 90 mP.

Results of Experiment 2

Attempts were made to measure P, V−H and V+H versus time for between 30seconds and 600 seconds. However, the reaction was immediate and thevalues obtained for P, V−H and V+H did not change over this time period.In particular, V−H was at 1.51 V, V+H was at 8.118, and V and P valueswere at 184 mP throughout the measurement period.

The results of Experiment 2 indicate that it was not possible to measurea kinetic curve in the absence of an additive. In particular, it was notpossible to obtain meaningful information during the measurement of thereaction between the fluorescently-labeled thyroxine and its specificantibody in the absence of benzoate.

Results of Experiment 3

Attempts were made to measure P, V−H and V+H versus time for between 30seconds and 600 seconds. However, the reaction was immediate and thevalues for P, V−H and V+H were constant over the measurement period. Inparticular, V−H was at 0.1580 V, V+H was at 5.243, and V and P valueswere at 30 mP throughout the measurement period.

The results of Experiment 3 indicate that it was not possible to obtainmeaningful information during the measurement of a kinetic curve inabsence of an additive due to non-specific binding. In particular, itwas not possible to follow the binding between F-thyroxine and itsspecific antibody, nor the inhibition of this binding by the unlabeledthyroxine in the absence of benzoate.

Results of Experiment 4

Polarization, V−H and V+H values were measured for between 10 and 600seconds. During this time course, a kinetic curve was obtained using ananti-thyroxine/F-thyroxine system, in which no unlabeled thyroxine wasadded. In particular, the polarization, V−H and V+H values increasedduring the 600 second time period.

The increase in polarization was due to the binding occurring betweenthe F-thyroxine and its specific antibody. The results of Experiment 4,which are provided in FIG. 1, indicate that it was possible to obtainmeaningful information during the measurement of a kinetic curve in thepresence of an additive.

Results of Experiment 5

A study of the competitive inhibition of the binding of the F-thyroxineto its specific antibody by unlabeled thyroxine in the presence ofsodium benzoate was performed.

The results of this competitive inhibition type assay of the presentinvention indicated that inhibition of the binding of F-thyroxine to itsspecific antibody by unlabeled thyroxine occurred. These results areshown in FIG. 1. In particular, in Experiment 5, a standard inhibitioncurve was obtained using various amounts of unlabeled thyroxine from 0μl to 20 μl using the protocol in Example 2. This standard curve is usedto measure unknown quantities of analyte. This also demonstrates it ispossible to perform an immunoassay in the presence of an additive.

Example 4 Effects of an Additive on Specific and Non-Specific Binding ina F-Thyroxine/Anti-Thyroxine System

With reference to FIG. 2, experiments were performed to determine theeffects of an additive on specific and non-specific binding. Theadditive used in the present example was sodium benzoate. The materialsdescribed in Example 1 and the measurement methodology described inExample 2 were used. In particular, 50 μl of human sera was added to3000 μl of Buffer 2 or PBS at pH 7.5 (no additive control) andthoroughly mixed by pipetting for 1 to 10 seconds. To this was added 0μl of unlabeled thyroxine and 0 μl, 20 μl or 50 μl of anti-sera stocksolution. The mixture was thoroughly mixed by pipetting for 1 to 10seconds. Then, 5 μl of the fluorescein-labeled thyroxine stock solutionwas added. This was followed by mixing for 1 to 10 seconds by pipetting.Then, d(V−H)/dt measurements were made versus time for between 10 and600 seconds. This protocol was followed for Experiments 6-9 in Table 2.This table shows the experimental set-ups, and the corresponding symbolsused in FIG. 2 for each experiment.

TABLE 2 Experiment Antiserum Human Sera F-thyroxine No. (μl) (μl) (μl)Additive 6 (♦) 0 50 5 + 7 (▪) 20 50 5 + 8 (▴) 50 50 5 + 9 (-X-) 20 50 5−Results

As shown in FIG. 2, it was only possible to measure an increase in PFID(V−H) values over the 600 second time course in the presence of anadditive (▴ and ▪). An increase in P and V+H values was also observedover this time course in the presence of the additive. V−H values didnot change in the absence of the additive (—X—; data not shown).

The data further show that the reaction only proceeds in the presence ofanti-sera (see constant V−H values for ♦, where no anti-sera waspresent). The overall results indicated that the additive inhibitsnon-specific binding, but does not inhibit specific binding.

Example 5 Kinetic Curve Measurable in the Presence of an Additive Usinga Rhodamine-Labeled Thyroxine/Anti-Thyroxine System

This example demonstrates the successful use of a non-fluorescein“red”-labeled thyroxine/anti-thyroxine system for fluorescencepolarization and polarized fluorescence intensity difference PFID (V−H)measurements as a substitute system for a fluorescein-labeledthyroxine/anti-thyroxine system. The protocol used is described below.The additive was sodium benzoate combined with an organic solvent (5%isopropanol).

-   -   1. Use clean glassware, washed with isopropanol.    -   2. Make a 0.5 M solution of sodium benzoate.    -   3. Make a solution of 5% isopropanol in 0.5 M sodium benzoate        (i.e., 2.5 ml 2-propanol in 47.5 mL 0.5 M aqueous sodium        benzoate). This will be named BUFFER.    -   4. 800 μl of BUFFER added to approximately 200 μg        5-carboxy-X-rhodamine-thyroxine (ROX-T4). This will be named        STOCK ROX-T4 solution.    -   5. 600 μl Buffer added to 1 mL curvette. The absorbance spectrum        is measured using a UV-VIS spectrophotometer, 400 nm to 800 nm.        This spectrum is used to define the absorbance of the background        as zero and to control that it is actually zero.    -   6. Add approximately 20 μl of STOCK ROX-T4 to cuvette and mix.        Measure the absorbance UV-VIS, 400 nm to 800 nm. Absorbance        maximum is measured to be 582 nm.    -   7. Using a rotating polarometer with a 75 watt Xenon short-arc        lamp. The rotating polarizer has three channels: Polarization        (P), Total Intensity (V+H), and V−H.    -   8. Add 2 μl STOCK ROX-T4 to 3000 μl (3 mL or 3 cc) BUFFER.        Measure P, V+H and V−H after short mixing and 30 second        equilibration. P=0.075; V+H=2291; V−H=178. Conclusions:        Intensity has increased due to presence of more fluorophore;        Polarization has remained constant because there is no binding.    -   9. Add 10 μl. Antibody solution (anti-thyroxine in PBS, dilution        unknown, probably 5 mg in 1000 μl PBS and further diluted 1:10        in PBS). New volume is 3017 μl. Measure P, V+H and V−H after        short mixing. Results are shown in Table 3 below:

TABLE 3 Time(s) P (mP) V + H V − H 0 75 2291 178 10 660 13 860 18 936 201006 26 1130 30 1175 33 1200 44 1259 49 1280 53 1290 62 392 70 395 781345 83 3375 95 397 600 398 3320 1340

These results are shown graphically in FIG. 3. The results indicate thatan increase in the values of P, V−H and V+H occurs over the 600 secondtime course of the reaction.

Since the reaction between the fluorescent conjugate and its antibody ismeasurable, a study of the inhibition of the binding of the fluorescentconjugate by a sample thought to contain an analyte of interest iscarried out. In particular, a change in the initial rate of polarizedfluorescence intensity difference PFID (V−H) is monitored as a functionof analyte in the sample.

The amount of analyte in the sample is determined by measuring thebinding of the fluorescent conjugate to the specific binding componentin the presence of the sample and determining the amount of analyte inthe sample from a standard curve. Such a standard curve is constructedby first measuring the binding of the fluorescent conjugate to itsspecific binding partner (e.g., its specific antibody) and determiningthe extent of inhibition of this binding by different known quantitiesof unlabeled analyte. The standard curve is then constructed, whichshows the degree of binding by the labeled analyte as a function of thequantity of the unlabeled analyte.

Example 6 Influence of Sodium Benzoate Combined with an Organic Solventon the Rate of the Reaction Between F-Thyroxine and Anti-Thyroxine andon the Non-Specific Binding of Serum Components

The present example shows the influence of sodium benzoate combined with10% 2-propanol on the velocity of the reaction between fluorescenthapten (F-thyroxine) and anti-thyroxine, and on the initial non-specificbinding between F-thyroxine and serum components. The buffer and stocksolutions described in Example 1 and the measurement methodologydescribed in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine (1 nM)    -   Anti-Thyroxine (1:300 dilution)    -   PBS with 10% 2-propanol    -   Benzoate in different concentrations    -   At t=0 s antibody was pipetted into the cuvette.    -   A 3 ml cuvette was used, the total volume in the cuvette was        2 ml. A small magnetic stirrer was used for mixing

In example 6, the reactions between F-Thyroxine and Anti-Thyroxine,which were carried out in the presence of serum under different reactionconditions, are compared (FIG. 4). There are seven different amounts ofsodium benzoate, between 0 and 1.0 Molar in the cuvette. The reactionwas performed in a phosphate buffered saline buffer containing 10%2-propanol. The table in FIG. 4 lists the two important parameters. Thefirst column shows the rate of change in polarization value after fiveseconds (t=5). The second column shows the polarization value before theaddition of antibody (t=0).

In reaction 6-1, the buffer background fluorescence was measured andblanked. F-thyroxine was added and mixed. The polarization value wasobserved to be 100 mP. Antibody (anti-T4) was added, stirred, and at t=5s the rate of change of P was observed to be 25 mP/s.

In reaction 6-2, the buffer contained an additional 5 μL serum. Thebackground fluorescence was measured and blanked. F-thyroxine was addedand mixed. The polarization value was observed to be 195 mP. Antibody(anti-T4) was added, stirred, and at t=5 s the rate of change of P wasobserved to be 9.7 mP/s.

The interaction between serum proteins and/or other serum components andF-thyroxine was quite strong, as the difference between the measured mPvalue before and after addition of serum almost doubled. The reactionrate between F-thyroxine and its antibody was also affected by thepresence of serum, as it slowed to less than half its value.

In experiments 6-3 through 6-7, the effect of increasing sodiumbenzoate, was be observed to perform two tasks. The first was to slowthe reaction rate down to about 30% its value from zero additive agent(9.7 to 3.2). The second was the elimination of non-specific binding, asexpressed by the decrease of the mP value due to the interaction withserum. This was reduced from 195 down to 100, equivalent to the originalmP value before the addition of serum as observed in experiment 6-1.

Example 7 Influence of Sodium Benzoate Combined with an Organic Solventon the Velocity of the Reaction Between F-Thyroxine and Anti-Thyroxinein the Absence of Non-Specific Binding Components

The present example shows the influence of sodium benzoate in theabsence of serum or other non-specific binding components, on theantibody reaction velocity between F-thyroxine and anti-T4 when thereaction was performed in a PBS buffer containing an additional 5%2-propanol (FIG. 5). The buffer and stock solutions described in Example1 and the measurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-thyroxine, 1 nM    -   Anti-Thyroxine (1:300)    -   PBS with 5% 2-propanol    -   Sodium Benzoate in different concentrations

From this experiment, it was observed that the reaction rate betweenF-thyroxine and antibody slowed dramatically as the concentration ofsodium benzoate increased to 1.0 molar.

Example 8 Influence of Sodium Benzoate Combined with an Organic Solventon the Velocity of the Reaction Between F-Thyroxine and Anti-Thyroxineand on Non-Specific Binding of BSA

The present example shows the influence of sodium benzoate in thepresence of the non-specific binding substance BSA, on the antibodyreaction velocity between F-thyroxine and anti-T4 and on thenon-specific binding between F-thyroxine and BSA (FIG. 6) when thereaction was performed in a PBS buffer containing an additional 5%2-propanol. The buffer and stock solutions described in Example 1 andthe measurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1 nM    -   Anti-Thyroxine (1:300)    -   PBS with 5% 2-propanol    -   BSA 0.1 μM    -   Sodium benzoate in different concentrations

It was observed that the non-specific binding due to the presence of BSAwas reduced by the addition of the additive, as seen by the lowering ofthe initial P value with respect to the measurement without theadditive. It was also observed that the rate of the antibody reactionwas substantially slowed by the addition increasing concentrations ofsodium benzoate.

Example 9 Influence of Guandine Hydrochloride Combined with an OrganicSolvent on the Velocity of the Reaction Between F-Thyroxine andAnti-Thyroxine and on Non-Specific Binding of BSA

The present example shows the influence of guanidine hydrochloride onthe antibody reaction velocity between F-thyroxine and anti-thyroxine inthe presence of the non-specific binding substance BSA and on thenon-specific binding between F-thyroxine and BSA (FIG. 7) when thereaction was performed in a PBS buffer containing an additional 5%2-propanol. The buffer and stock solutions described in Example 1 andthe measurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1 nM    -   Anti-Thyroxine (1:300)    -   PBS with 5% 2-propanol    -   BSA 0.1 μM    -   Guanidine hydrochloride in different concentrations

It was observed that the non-specific binding due to the presence of BSAwas reduced by the addition of the additive, as seen by the lowering ofthe initial P valve with respect to the measurement without theadditive. It was also observed that the rate of the reaction wassubstantially slowed by the addition of increasing concentrations ofguanidine hydrochloride.

Example 10 Influence of Sodium P-Tolulene Sulfonate Combined with anOrganic Solvent on the Velocity of the Reaction Between F-Thyroxine andAnti-Thyroxine and on Non-Specific Binding of BSA

The present example shows the influence of sodium p-toluene sulfonate onthe antibody reaction velocity between F-thyroxine and anti-thyroxine inthe presence of the non-specific binding substance BSA and on thenon-specific binding between F-thyroxine and BSA (FIG. 8) when thereaction was performed in a PBS buffer containing an additional 5%2-propanol. The buffer and stock solutions described in Example 1 andthe measurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1 nM    -   Anti-Thyroxine (1:300)    -   PBS with 5% 2-propanol    -   BSA 0.1 μM    -   Sodium p-toluene sulfonate in different concentrations

It was observed that the non-specific binding due to the presence of BSAwas reduced by the addition of the additive, as seen by the lowering ofthe initial P value with respect to the measurement without theadditive. It was also observed that the rate of the reaction wassubstantially slowed by the addition of increasing concentrations ofsodium p-tolulene sulfonate.

Example 11 Influence of Potassium Benzoate Combined with an OrganicSolvent on the Velocity of the Reaction Between F-Thyroxine andAnti-Thyroxine and on Non-Specific Binding of BSA

The present example shows the influence of potassium benzoate on theantibody reaction velocity between F-thyroxine and anti-thyroxine in thepresence of the non-specific binding substance BSA and on thenon-specific binding between F-thyroxine and BSA (FIG. 9) when thereaction was performed in a PBS buffer containing an additional 4.75%2-propanol. The buffer and stock solutions described in Example 1 andthe measurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1 nM    -   Anti-Thyroxine (1:300)    -   PBS with 4.75% 2-propanol    -   BSA 0.1 μM    -   Potassium benzoate in different concentrations

It was observed that the non-specific binding due to the presence of theBSA was reduced by the addition of the additive, as seen by the loweringof the initial P value with respect to the measurement without theadditive. It was also observed that the rate of the reaction wassubstantially slowed by the addition of increasing concentrations ofpotassium benzoate.

Example 12 Influence of Sodium Chloroacetate Combined with an OrganicSolvent on the Velocity of the Reaction Between F-Thyroxine andAnti-Thyroxine and on Non-Specific Binding of BSA

The present example shows the influence of sodium chloroacetate on theantibody reaction velocity between F-thyroxine and anti-thyroxine in thepresence of the non-specific binding substance BSA and on thenon-specific binding between F-thyroxine and BSA (FIG. 10) when thereaction was performed in a PBS buffer containing an additional 4.75%2-propanol. The buffer and stock solutions described in Example 1 andthe measurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1 nM    -   Anti-Thyroxine (1:300)    -   PBS with 4.75% 2-propanol    -   BSA 0.1 μM    -   Sodium chloroacetate in different concentrations

It was observed that the non-specific binding due to the presence of BSAwas reduced by the addition of the additive. It was also observed thatthe rate of the reaction was substantially slowed by the addition ofincreasing concentrations of sodium chloroacetate.

Example 13 Influence of Sodium Dichloroacetate Combined with an OrganicSolvent on the Velocity of the Reaction Between F-Thyroxine andAnti-Thyroxine and on Non-Specific Binding of BSA

The present example shows the influence of sodium dichloroacetate on theantibody reaction velocity between F-thyroxine and anti-thyroxine in thepresence of the non-specific binding between F-thyroxine and BSA (FIG.11) when the reaction was performed in a PBS buffer containing anadditional 4.75% 2-propanol. The buffer and stock solutions described inExample 1 and the measurement methodology described in Example 2 wereused.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1 nM    -   Anti-Thyroxine (1:300)    -   PBS with 4.75% 2-propanol    -   BSA 0.1 μM    -   Sodium dichloroacetate in different concentrations

It was observed that the non-specific binding due to the presence of BSAwas reduced by the addition of the additive, as seen by the lowering ofthe initial P valve with respect to the measurement without theadditive. It was also observed that the rate of the reaction wassubstantially slowed by the addition of increasing concentrations ofsodium dichloroacetate.

Example 14 Influence of Sodium Salicylate Combined with an OrganicSolvent on the Velocity of the Reaction Between F-Thyroxine andAnti-Thyroxine and on Non-Specific Binding of BSA

The present example shows the influence of sodium salicylate on theantibody reaction velocity between F-thyroxine and anti-thyroxine in thepresence of the non-specific binding substance BSA and on thenon-specific binding between F-thyroxine and BSA (FIG. 12) when thereaction was performed in a PBS buffer containing an additional 4.75%2-propanol. The buffer and stock solutions described in Example 1 andthe measurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1.5 nM    -   Anti-Thyroxine (1:300)    -   PBS with 4.75% 2-propanol    -   BSA 0.1 μM    -   Sodium Salicylate in different concentrations

It was observed that the non-specific binding due to the presence of BSAwas reduced by the addition of the additive, as seen by the lowering ofthe initial P valve with respect to the measurement without theadditive. It was also observed that the rate of the reaction wassubstantially slowed by the addition of increasing concentrations ofsodium salicylate.

Example 15 Influence of N-hydroxysuccinimide Combined with an OrganicSolvent on the Velocity of the Reaction Between F-Thyroxine andAnti-Thyroxine and on Non-Specific Binding of BSA

The present example shows the influence of N-hydroxysuccinimide on theantibody reaction velocity between F-thyroxine and anti-thyroxine in thepresence of the non-specific binding substance BSA and on thenon-binding specific binding between F-thyroxine and BSA (FIG. 13) whenthe reaction was performed in a PBS buffer containing an additional4.75% 2-propanol and unspecific binding of fluorophore and BSA (FIG.13). The buffer and stock solutions described in Example 1 and themeasurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1.5 nM    -   Anti-Thyroxine (1:300)    -   PBS with 4.75% 2-propanol    -   BSA 0.1 μM    -   N-Hydroxysuccinimide in different concentrations

The ΔP diagram shows the difference of the P value of the freefluorescence labelled compound and its P value during the reaction,where ΔP=P−P_(t=0). This shows the increase of the P value due only tothe binding of analyte and antibody. It was observed that thenon-specific binding due to the presence of BSA was slightly reduced bythe addition of the additive. It was also observed that the rate of thereaction was substantially slowed by the addition of increasingconcentrations of N-hydroxysuccinimide.

Example 16 Influence of Sodium Chloride Combined with an Organic Solventon the Velocity of the Reaction Between F-Thyroxine and Anti-Thyroxineand on Non-Specific Binding of BSA

The present example shows the influence of sodium chloride on theantibody reaction velocity between F-thyroxine and anti-thyroxine in thepresence of the non-specific binding substance BSA and on thenon-specific binding between F-thyroxine and BSA (FIG. 14) when thereaction was performed in a PBS buffer containing an additional 4.75%2-propanol. The buffer and stock solutions described in Example 1 andthe measurement methodology described in Example 2 were used.

Experimental Conditions:

-   -   Fluorescein-Thyroxine, 1.5 nM    -   Anti-Thyroxine (1:300)    -   PBS with 4.75% 2-propanol    -   BSA 0.1 μM    -   Sodium Chloride in different concentrations

It was observed that the non-specific binding due to the presence of BSAwas reduced by the addition of the additive. However, it was alsoobserved that the rate of the reaction was not substantially slowed bythe addition of increasing concentrations of sodium chloride therebymaking the antibody reaction more difficult to study in the presence ofsodium chloride relative to in the presence of other salt agents.

Example 17 Effects of Sodium Benzoate Combined with an Organic Solventon the Velocity of the Reaction Between Rhodamine-Thyroxine andAnti-Thyroxine and on Non-Specific Binding of Serum Components

The present example shows the effects in a Rhodamine-Thyroxine/AntiThyroxine system of different amounts of sodium benzoate in serum and ata higher concentration of alcohol (FIG. 15). The serum was diluted 1:500in PBS/10% 2-propanol The buffer and stock solutions, as well as themeasurement methodology described in Example 5 were used.

Experimental Conditions:

-   -   Rox-Thyroxine, 1.5 nM    -   Anti-Thyroxine (1:300)    -   PBS with 10% 2-propanol    -   Serum 1:500    -   Sodium benzoate in different concentrations

It was observed that the non-specific binding due to the presence ofserum components was substantially reduced by the addition of thiscombination of additives. It was also observed that the rate of thereaction was substantially slowed by the addition of increasingconcentrations of sodium benzoate.

Example 18 Standard Curve Constructed by Monitoring for a Change in theInitial Rate of Polarization as a Function of Different Known Quantitiesof the Analyte Thyroxine

The present example is directed to an experiment, such as in Example 17,performed in serum, and shows that the antibody reaction betweenRox-thyroxine and anti-thyroxine can be performed in the presence ofadditive (FIG. 16). In particular, FIG. 16 shows a calibration curve,measured in the following conditions:

-   -   1500 μl Serum diluted 1:500 with (PBS buffer containing 10%        2-propanol and 0.7 M sodium benzoate)    -   1.5 nM Rox-Thyroxine and 20 μl anti-Thyroxine (1:300 dilution)        The x-axis of the graph in FIG. 16 is the concentration (nM) of        analyte thyroxine in the cuvette. From the standard curve, it is        possible to measure unknown quantities of the analyte.

What is claimed is:
 1. A method for reducing non-specific binding in anassay, comprising: (a) providing a reaction mixture, which comprises oris suspected to comprise a first component and a second componentcapable of binding to each other in a specific binding reaction; (b)adding non-physiological amounts of at least one additive to thereaction mixture before, during or after binding in a sufficient amountto reduce non-specific binding in the reaction mixture; and (c)monitoring or measuring the presence and/or concentration of at leastone of said first and second components after step (b).
 2. The method ofclaim 1, wherein the additive is present in an amount of about 0.2 M toabout 2.5 M in the assay.
 3. The method of claim 1, wherein the additiveis present in an amount of about 5% to about 20% (weight/volume) of thereaction mixture.
 4. The method of claim 1, wherein the additive is asalt of an anion selected from the group consisting of salicylate,trichloroacetate, thiocyanate, perchlorate and benzoate.
 5. The methodof claim 1, wherein the additive is selected from the group consistingof 8-anilino-1-napthalene-sulfonic acid, 2-Guanidinobenzimidazole,2,3,5-triacetylguanosine, Benzimidazolylurea, acetamide,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), sodiumtrichloroacetate, sodium deoxycholate, creatine benzimidazole, sodiump-toluene-sulfonate, sodium dichloro acetate, sodium iodide, sodiumfluoride, sodium chloroacetate, 5-benzimidazolecarboxylic acid,Salicylamide, guanidine hydrochloride, sodium chloride,2-benzimidazole-proprionic acid, 2-benzimidazolemethanol, Sodiumchlorodifluoroacetate, 4-guanidinobenzoic acid, 3-chlorobenzoic acid,N-hydroxy succinimide, guanidine and Potassium benzoate, organicsolvents and combinations thereof.
 6. The method of claim 1, wherein theadditive affects the order of reaction with respect to the firstcomponent and the second component.
 7. The method of claim 1, whereinthe additive is present in a sufficient amount to make the bindingreaction first order with respect to the concentration of each of thefirst and second components.
 8. The method of claim 1, wherein theadditive reduces non-specific binding between molecules of the firstcomponent.
 9. The method of claim 1, wherein the additive reducesnon-specific binding between molecules of the second component.
 10. Themethod of claim 1, wherein the additive reduces non-specific bindingbetween an interfering substance in the reaction mixture and at leastone of the first and second components.
 11. The method of claim 1,wherein the assay is selected from the group consisting of fluorescencepolarization assays, fluorescence intensity assays, enzyme immunoassays,gold-colloidal assays and latex-bead assays.
 12. The method of claim 11,wherein the fluorescence intensity assay is a Polarized FluorescenceIntensity Difference (PFID) assay.
 13. The method of claim 1, wherein atleast one of the first and second components is fluorescently-labeled.14. A method for reducing non-specific binding in a fluorescence assayfor the detection and/or measurement of an analyte in a sample,comprising: (a) providing a fluorescent conjugate of the analyte; (b)providing a component that specifically binds to the analyte and itsfluorescent conjugate; (c) combining the fluorescent conjugate and thespecific binding component with a sample which comprises or is suspectedto comprise the analyte under conditions suitable for the specificbinding component to specifically bind to the analyte and itsfluorescent conjugate; (d) adding non-physiological amounts of at leastone additive to the assay before, during or after the combining step toreduce non-specific binding in the assay; and (e) monitoring for theinhibition of the binding of the fluorescent conjugate to the specificbinding component by the sample.
 15. The method of claim 14, wherein theadditive is present in an amount of about 0.2 M to about 2.5 M in theassay.
 16. The method of claim 14, wherein the additive is present in anamount of about 5% to about 20% by weight/volume in the assay.
 17. Themethod of claim 14, wherein the additive is a salt of an anion selectedfrom the group consisting of chloride, salicylate, trichloroacetate,thiocyanate, perchlorate and benzoate.
 18. The method of claim 14,wherein the additive is selected from the group consisting of8-anilino-1-napthalene-sulfonic acid, 2-Guanidinobenzimidazole,2,3,5-triacetylguanosine, Benzimidazolylurea, acetamide,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), sodiumtrichloroacetate, sodium deoxycholate, creatine benzimidazole, sodiump-toluene-sulfonate, sodium dichloro acetate, sodium iodide, sodiumfluoride, sodium chloroacetate, 5-benzimidazolecarboxylic acid,Salicylamide, guanidine hydrochloride, sodium chloride,2-benzimidazole-proprionic acid, 2-benzimidazolemethanol, Sodiumchlorodifluoroacetate, 4-guanidinobenzoic acid, 3-chlorobenzoic acid,N-hydroxy succinimide, guanidine and Potassium benzoate, organicsolvents and combinations thereof.
 19. The method of claim 14, whereinthe additive affects the order of reaction with respect to the specificbinding component and the analyte.
 20. The method of claim 14, whereinthe additive reduces non-specific binding between molecules of thespecific binding component.
 21. The method of claim 14, wherein theadditive reduces non-specific binding between molecules of the analyte.22. The method of claim 14, wherein the additive reduces non-specificbinding between an interfering substance in the assay and at least oneof the specific binding component and the analyte.
 23. The method ofclaim 14, wherein the assay is selected from the group consisting of afluorescence polarization assay, a fluorescence-based enzyme-linkedimmunosorbent assay (ELISA) and a Polarized Fluorescence IntensityDifference (PFID) assay.
 24. The method of claim 23, wherein themonitoring step comprises monitoring for a change in the initial rate ofpolarization as a function of an amount of analyte in the sample. 25.The method of claim 14, wherein the monitoring step comprises measuringthe binding of the fluorescent conjugate to the specific bindingcomponent; and determining the extent of inhibition of this binding bydifferent known quantities of unlabeled analyte.
 26. The method of claim25, wherein the monitoring step further comprises constructing astandard curve which shows the degree of binding by the fluorescentconjugate as a function of the quantity of the unlabeled analyte. 27.The method of claim 26, wherein the monitoring step further comprisesdetermining the amount of analyte in the sample by measuring the bindingof the fluorescent conjugate to the specific binding component in thepresence of the sample and determining the amount of analyte in thesample from the standard curve.
 28. The method of claim 14, wherein theanalyte is an organic contaminant.
 29. The method of claim 14, whereinthe organic contaminant is of environmental concern.
 30. The method ofclaim 29, wherein the organic contaminant is a fungal or microbialtoxin.
 31. The method of claim 14, wherein the analyte is a drug. 32.The method of claim 14, wherein the analyte is a steroid or hormone. 33.The method of claim 14, wherein the analyte is a protein or peptide. 34.The method of claim 14, wherein the analyte is a lipid or sugar.
 35. Themethod of claim 14, wherein the specific binding component is anantibody.
 36. The method of claim 14, wherein the specific bindingcomponent is a receptor.
 37. The method of claim 14, wherein thespecific binding component is on a solid substrate.
 38. The method ofclaim 37, wherein the solid substrate is selected from the groupconsisting of glass, plastic and paper.
 39. The method of claim 14,wherein the fluorescent conjugate comprises a fluorescent dye.
 40. Themethod of claim 39, wherein the fluorescent dye is selected from thegroup consisting of fluorescein, rhodamine and derivatives thereof. 41.The method of claim 14, wherein the fluorescent conjugate comprisesthyroxine.
 42. The method of claim 41, wherein the specific bindingcomponent is anti-thyroxine.
 43. The method of claim 14, wherein the atleast one additive is a combination of a salt agent and an organicsolvent.